Directed switch-mediated DNA recombination

ABSTRACT

Switch regions derived from an immunoglobulin (Ig) gene are used to direct recombination between a targeting construct containing a promoter, a switch region (S 1 ), and 2) a target locus minimally containing a promoter, a switch region (S 2 ), and a target sequence.

FIELD OF THE INVENTION

[0001] This invention relates generally to methods and compositions foruse in recombinant DNA technology, particularly in. methods formanipulation of DNA sequences encoding antibodies, proteins, or portionsthereof.

BACKGROUND OF THE INVENTION

[0002] The basic immunoglobulin (Ig) structural unit in vertebratesystems is composed of two identical “light” polypeptide chains(approximately 23 kDa), and two identical “heavy” chains (approximately53 to 70 kDa). The four chains are joined by disulfide bonds in a “Y”configuration, and the “tail” portions of the two heavy chains are boundby covalent disulfide linkages when the immunoglobulins are generatedeither by B cell hybridomas or other cell types.

[0003] A schematic of the general antibody structure is shown in FIG. 1.The light and heavy chains are each composed of a variable region at theN-terminal end, and a constant region at the C-terminal end. In thelight chain, the variable region (termed “V_(L)J_(L)”) is composed of avariable (V_(L)) region connected through the joining (J_(L)) region tothe constant region (C_(L)). In the heavy chain, the variable region(V_(H)D_(H)J_(H)) is composed of a variable (V_(H)) region linkedthrough a combination of the diversity (D_(H)) region and the joining(J_(H)) region to the constant region (C_(H)). The V_(L)J_(L) andV_(H)D_(H)J_(H) regions of the light and heavy chains, respectively, areassociated at the tips of the Y to form the antibody's antigen bindingportion and determine antigen binding specificity.

[0004] The (C_(H)) region defines the antibody's isotype, i.e., itsclass or subclass. Antibodies of different isotypes differ significantlyin their effector functions, such as the ability to activate complement,bind to specific receptors (e.g., Fc receptors) present on a widevariety of cell types, cross mucosal and placental barriers, and formpolymers of the basic four-chain IgG molecule.

[0005] Antibodies are categorized into “classes” according to the C_(H)type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, orIgA). There are at least five types of C_(H) genes (Cμ, Cγ, Cδ, Cε, andCα), and some species (including humans) have multiple C_(H) subtypes(e.g., Cγ₁,Cγ₂, Cγ₃, and Cγ₄ in humans). There are a total of nine C_(H)genes in the haploid genome of humans, eight in mouse and rat, andseveral fewer in many other species. In contrast, there are normallyonly two types of light chain constant regions (C_(L)), kappa (κ) andlambda (λ), and only one of these constant regions is present in asingle light chain protein (i.e., there is only one possible light chainconstant region for every V_(L)J_(L) produced). Each heavy chain classcan be associated with either of the light chain classes (e.g., a C_(H)γ region can be present in the same antibody as either a κ or λ lightchain), although the constant regions of the heavy and light chainswithin a particular class do not vary with antigen specificity (e.g., anIgG antibody always has a Cγ heavy chain constant region regardless ofthe antibody's antigen specificity).

[0006] Each of the V, D, J, and C regions of the heavy and light chainsare encoded by distinct genomic sequences. Antibody diversity isgenerated by recombination between the different V_(H), D_(H), and J_(H)gene segments in the heavy chain, and V_(L) and J_(L) gene segments inthe light chain. The recombination of the different V_(H), D_(H), andJ_(H) genes is accomplished by DNA recombination during B celldifferentiation. Briefly, the heavy chain sequence recombines first togenerate a D_(H)J_(H) complex, and then a second recombinatorial eventproduces a V_(H)D_(H)J_(H) complex. A functional heavy chain is producedupon transcription followed by splicing of the RNA transcript.Production of a functional heavy chain triggers recombination in thelight chain sequences to produce a rearranged V_(L)J_(L) region which inturn forms a functional V_(L)J_(L)V_(L) region, i.e., the functionallight chain.

[0007] During the course of B cell differentiation, progeny of a singleB cell can switch the expressed immunoglobulin isotype from IgM to IgGor other classes of immunoglobulin without changing the antigenspecificity determined by the variable region. This phenomenon, known asimmunoglobulin class-switching, is accompanied by DNA rearrangement thattakes place between switch (S) regions located 5′ to each C_(H) gene(except for Cγ) (reviewed in Honjo (1983) Annu. Rev. Immunol. 1:499-528,and Shimizu & Honjo (1984) Cell 36:801-803). S-S recombination bringsthe V_(H)D_(H)J_(H) exon to the proximity of the C_(H) gene to beexpressed by deletion of intervening C_(H) genes located on the samechromosome. The class-switching mechanism is directed by cytokines(Mills et al. (1995) J. Immunol. 155:3021-3036). Switch regions vary insize from 1 kb (Sε) to 10 kb (Sγ₁), and are composed of tandem repeatsthat vary both in length and sequence (Gritzmacher (1989) Crit. Rev.Immunol. 9:173-200). Several switch regions have been characterizedincluding the murine Sμ, Sε, Sα, Sγ3, Sγ1, Sγ2b and Sγ2a switch regionsand the human Sμ switch region (Mills et al. (1995) supra; Nikaido etal. (1981) Nature 292:845-8; Marcu et al. (1982) Nature 298:87-89;Takahashi et al. (1982) Cell 29:671-9; Mills et al. (1990) Nucleic AcidsRes. 18:7305-16; Nikaido et al. (1982) J. Biol. Chem. 257:7322-29;Stanton et al. (1982) Nucleic Acids Res. 10:5993-6006; Gritzmacher(1989) supra; Davis et al. (1980) Science 209:1360; Obata et al. (1981)Proc. Natl. Acad. Sci. U.S.A. 78:2437-41; Kataoka et al. (1981) Cell23:357; Mowatt et al. (1986) J. Immunol. 136:2674-83; Szurek et al.(1985) J. Immunol. 135:620-6; and Wu et al. (1984) EMBO J. 3:2033-40).

[0008] Observations that a single B cell can express more than oneisotype simultaneously on its surface is not explained by theclass-switching mechanism since S-S recombination is limited tointrachromosomal recombination and results in deletion of the exchangedC_(H)gene. A second mechanism, called trans-splicing, has been describedin which two transcripts generated from different chromosomes are joinedto form a single continuous transcript (Shimizu et al. (1991) J. Exp.Med. 173:1385-1393). Transgenic mice carrying a rearranged expressibleV_(H)D_(H)J_(H) heavy chain μ gene integrated outside the mouse IgHlocus were found to produce mRNA having the V_(H)D_(H)J_(H) region ofthe transgene correctly spliced to the endogenous C_(H) region. As withS-S recombination, the frequency of trans-splicing is low, and thefactors regulating both mechanisms are not well understood.

[0009] The value and potential of antibodies as diagnostic andtherapeutic reagents has been long-recognized in the art. Unfortunately,the field has been hampered by the slow, tedious processes required toproduce large quantities of an antibody of a desired specificity. Theclassical cell fusion techniques allowed for efficient production ofmonoclonal antibodies by fusing the B cell producing the antibody withan immortalized cell line. The resulting cell line is called a hybridomacell line. However, most of these monoclonal antibodies are produced inmurine systems and are recognized as “foreign” proteins by the humanimmune system. Thus the patient's immune system elicits a responseagainst the antibodies, which results in antibody neutralization andclearance, and/or potentially serious side-effects associated with theanti-antibody immune response.

[0010] One approach to this problem has been to develop human or“humanized” monoclonal antibodies, which are not as easily “recognized”as foreign epitopes, and avoid an anti-antibody immune response in thepatient. Applications of human B cell hybridoma-produced monoclonalantibodies have promising potential in the treatment of cancer,microbial, and viral infections, B cell immunodeficiencies associatedwith abnormally low antibody production, autoimmune diseases,inflammation, transplant rejection and other disorders of the immunesystem, and other diseases. However, several obstacles remain in thedevelopment of such human monoclonal antibodies. For example, many humantumor antigens may not be immunogenic in humans and thus it may bedifficult to isolate human B cells producing antibodies against humanantigens.

[0011] Attempts to address the problems associated with antibodies forhuman therapeutics have used recombinant DNA techniques. Most of theseefforts have focused on the production of chimeric antibodies having ahuman C_(H) region and non-human (e.g., murine) antigen combining(variable) regions. These chimeric antibodies are generally produced bycloning the desired antibody variable region and/or constant region,combining the cloned sequences into a single construct encoding all or aportion of a functional chimeric antibody having the desired variableand constant regions, introducing the construct into a cell capable ofexpressing antibodies, and selecting cells that stably express thechimeric antibody. Alternatively, the chimeric antibody is produced bycloning the desired variable region or constant region, introducing theconstruct into an antibody-producing cell, and selecting for chimericantibody-producing cells that result from homologous recombinationbetween the desired variable region and the endogenous variable region,or the desired constant region and the endogenous constant region.Examples of techniques which rely upon recombinant DNA techniques suchas those described above to produce chimeric antibodies are described inPCT Publication No. WO 86/101533 (Neuberger et al.), and in U.S. Pat.Nos. 4,816,567 (Cabilly et al.) and 5,202,238 (Fell et al.). Thesemethods require transferring DNA from one cell to another, thus removingit from its natural locus, and thus require careful in vitromanipulation of the DNA to ensure that the final antibody-encodingconstruct is functional (e.g., is capable of transcription andtranslation of the desired gene product).

[0012] There is a clear need in the field for a method for producing adesired protein or antibody which does not require multiple cloningsteps, in more efficient than conventional homologous recombination, andcan be carried out in hybridoma cells.

SUMMARY OF THE INVENTION

[0013] The present invention features a method of replacing one DNAsequence with another using switch (S) regions derived from animmunoglobulin (Ig) gene. The method of the invention allows any twopieces of DNA to be “switched” or a piece of exogenous DNA to beinserted into a site containing a natural or artificial S region. Thusthe method of the invention allows directed recombination to occur andeliminates many cloning steps required by current recombinant DNAmethods.

[0014] In the method of the invention, directed recombination is broughtabout between a targeting construct and a target locus. The nucleic acidtargeting construct is composed minimally of a switch region and apromoter operably linked to and 5′ of the switch region. Additionally,depending on the desired recombinatorial product, the targetingconstruct can also contain a modifying sequence operably linked to and3′ of the switch region, and other DNA sequences between the promoterand switch regions, e.g., 5′ of the switch region and 3′ of the promoterregion. Of particular interest is the use of a targeting construct withan Ig heavy chain to facilitate isotype switching, e.g., replacement ofan endogenous constant region (C_(H)) in an antibody heavy chain gene(target sequence) with a C_(H) of a different subtype, isotype, orspecies of origin (modifying sequence). For example, exogenous DNAencoding the constant or variable region of an antibody light or heavychain can be switched with the constant or variable region of anendogenous sequence to create a sequence which encodes an antibody witha different constant or variable region. In a broader sense, the methodof the invention is widely applicable to manipulate DNA sequences forproduction of a desired protein or protein component, including theproduction of chimeric antibodies having a desired variable regionlinked to a non-antibody polypeptide (e.g., a detectable polypeptidelabel, or a polypeptide having a desired activity).

[0015] In one aspect, the invention features a method for directedswitch-mediated recombination by a) introducing a targeting constructinto a cell having a target locus, the target locus being minimallycomposed of a promoter, a switch region, and a target sequence, whereinthe targeting construct is minimally composed of a promoter and a switchregion, and can contain additional modifying sequences, b) culturing thecell to allow transcription of the target locus and the targetingconstruct, thereby promoting recombination of the switch regions of thetarget locus and the targeting construct, and c) selecting a cellcontaining the desired recombined DNA product sequence, minimallycomposed of a switch region (composed of DNA sequences from one or boththe target locus switch region and targeting construct switch region).

[0016] In a specific embodiment of the invention, the targetingconstruct (P₁-S₁) is composed of a promoter (P₁) and switch region (S₁)and the target locus (P₂-S₂-T) is composed of a promoter (P₂), anaturally occurring or artificially inserted switch region (S₂), and atarget sequence (T). Directed S-S recombination between the S-S regionsresults in a DNA sequence having the P₁ promoter of the targetingconstruct, a switch region containing DNA sequences from one or both S₁and S₂ regions, and the T sequence (P₁-S₁/S₂-T). In this embodiment, thetarget sequence is removed from the control of the target locus promoterand placed under control of the desired P₁ promoter. Cells containingthe desired DNA sequence are selected by methods known in the art,including Southern blot analysis or PCR.

[0017] In another embodiment, the targeting construct (P₁-S₁-M) iscomposed of a promoter (P₁), a switch region (S₁), and a modifyingsequence (M), and the target locus (P₂-S₂-T) is composed of a promoter(P₂), a naturally occurring or artificially inserted switch region (S₂),and a target sequence (T). Directed S-S recombination between the S-Sregions results in two possible recombinatorial product sequences, onehaving the P₁ promoter of the targeting construct, a switch regioncontaining DNA sequences from one or both S₁ and S₂ regions, and the Tsequence (P₁-S₁/S₂-T), and a second sequence having a P₂ promoter, aswitch region-containing DNA sequences from one or both S₁ and S₂regions, and the M sequence (P₁-S₁/S₂-M). In this embodiment, cellsexpressing the M sequence are selected by methods known in the art,including Southern or Northern blot analysis.

[0018] In a third embodiment, the targeting construct (P₁-Z-S₁) iscomposed of a promoter (P₁), DNA sequences 5′ to the switch region (Z),and the switch region (S₁). The target locus (P₂-S₂-T) is composed of apromoter (P₂), a naturally occurring or artificially inserted switchregion (S₂), and a target sequence (T). Directed S-S recombinationbetween the switch regions results in a DNA sequence having the P₁promoter of the targeting construct, the Z DNA sequences, a switchregion containing DNA sequences from one or both switch regions, and theT sequence (P₁-Z-S₁/S₂-T).

[0019] The target locus is a DNA sequence having a switch region, andmay be a native, naturally-occurring sequence (e.g., an Ig locus of anantibody-producing cell), a rearranged Ig locus, or a recombinantlyproduced DNA sequence artificially inserted at a desired site. Thetarget locus. can be either an extrachromosomal element or a stablyintegrated chromosomal element. Preferably, the target locus encodes anantibody heavy chain gene. The targeting construct is either anextrachromosomal element or a stably integrated chromosomal element.Where the target locus is an antibody heavy chain gene, the modifyingsequence of the targeting construct preferably encodes a different ormodified heavy chain constant region or a non-antibody sequence ofinterest (e.g., a detectable polypeptide label, an enzyme, a toxin, or agrowth factor).

[0020] The invention provides a method of modifying a DNA sequence bydirected S-S recombination. The invention allows DNA recombination to bedirected to any site which contains a naturally-occurring switch regionor synthetic switch region, including a site into which an S region hasbeen artificially inserted.

[0021] The invention provides a method to replace or modify a first DNAsequence (a target sequence) with a second DNA sequence (a modifyingsequence) without the need for isolating the nucleotide sequencecontaining the target sequence, excising the target sequence, andligating the modifying sequence in place of the target sequence. Theinvention also provides a method to replace portions of apolypeptide-encoding sequences with a heterologous amino acid sequence,where the polypeptide is composed of two distinct components (e.g., anN-terminal component and a C-terminal component) that, for example,confer distinct functional or structural characteristics upon thepolypeptide (e.g., ligand binding or cell-binding). For example, theinvention allows for the substitution of either the N-terminal portionwith a different, heterologous amino acid-encoding sequence, or theC-terminal portion with a different, heterologous amino acid-encodingsequence.

[0022] Directed switch-mediated recombination allows recombination tooccur at a specific, pre-selected region with an increased efficiencyrelative to the naturally-occurring mechanism which is limited to theimmunoglobulin heavy chain. The method of the invention removesswitch-mediated recombination from the limitations of its normalregulatory environment, allowing recombination to be controlled asneeded with, for example, the use of constitutive or induciblepromoters.

[0023] The ability to accomplish directed in vitro S-mediatedrecombination avoids tedious, time-consuming manipulation of DNA usingconventional recombinant DNA techniques while providing a highlyefficient method of inserting a DNA sequence. For example, the methodallows the detectable label portion of fusion proteins (e.g.,β-galactosidase) to be readily exchanged for a different amino acidsequence (e.g., alkaline phosphatase).

[0024] In a specific application of the method of the invention,directed S-S recombination is used to replace the constant region of anantibody heavy chain gene with a different or modified constant regionwithout the need for extensive manipulation of the antibody heavy chaingene. Additionally, the method of the invention allows the antibody geneto be maintained in its native locus.

[0025] These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the compositions, composition components, methodsand method steps of the invention as set forth below.

BRIEF DESCRIPTION OF THE DRAWING

[0026]FIG. 1 is a schematic showing the basic immunoglobulin structure.

[0027]FIG. 2A is a schematic showing the basic components of a targetlocus consisting of a promoter (P₂), switch region (S₂), and a targetsequence (T).

[0028]FIG. 2B is a schematic showing the basic components of a targetlocus consisting of a promoter (P₂) DNA sequences positioned 3′ of thepromoter and 5′ of the switch region (Y), switch region (S₂), and atarget sequence (T).

[0029]FIG. 3A is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁) and switch region (S₁).

[0030]FIG. 3B is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁), switch region (S₁), andmodifying sequences.

[0031]FIG. 3C is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁) DNA sequences positioned 3′ ofthe promoter and 5′ of the switch region (Z), and switch region (S₁).

[0032]FIG. 3D is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁), DNA sequences positioned 3′ ofthe promoter and 5′ of the switch region (Z), switch region (S₁), andmodifying sequences, which may include additional components such as aselectable marker gene and/or an amplification gene.

[0033]FIG. 4A is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁-S₁ and target locus P₂-S₂-T.

[0034]FIG. 4B is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁-S₁-M and target locus P₂-S₂-T.

[0035]FIG. 4C is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁-Z-S₁ and target locus P₂-S₂-T.

[0036]FIG. 5 is a schematic of a directed targeting construct of theinvention (pTSW-1.4) having the entire 23 kb human γ2 locus (5′ controlelements, I exon, switch regions, coding sequences, membrane andsecretory exons, polyA), souse 3′ enhancer sequence, CMVpromoter/enhancer cassette, and SV2 hygromycin selectable marker.

[0037]FIG. 6 is a schematic of a targeting construct of the invention(pTSW-1.9) with the elements as described in the legend to FIG. 5, withthe CMV promoter/enhancer cassette in the opposite orientation to thatof pTSW-1.4.

[0038]FIG. 7 is a schematic of a targeting construct of the invention(pTSW-2) having a 12 kb BamHI fragment cloned from the 23 kb human γ2germline clone (including switch regions and the human γ2 open readingframe; the I exon and 5′ control elements are not included), CMVpromoter/enhancer cassette providing splice donor site, and SV2hygromycin selectable marker; the mouse 3′ enhancer is not included.

[0039]FIG. 8 is a schematic of a targeting construct of the invention(pTSW-3.1) having 10 kb of cloned HindIII-EcoRI mouse γ1 genomic switchfragment (5′ control elements, I exon, and mouse γ1 switch sequences),CMV promoter cassette (SFFV promoter cassette in the pTSW-3.2 series),genomic clone of human γ2 open reading frame and splice acceptor, SV2hygromycin selectable marker, and optionally the mouse 3′ enhancersequence.

[0040]FIG. 9 is a schematic of a targeting construct of the invention(pTSW-3.1BglII) having 7.9 kb BglII-EcoRI mouse γ1genomic switchfragment (I exon and mouse γ1 switch sequences; 5′ control elements notincluded), CMV promoter cassette (SFFV promoter cassette in the pTSW-3.2series), genomic clone of human γ2 open reading frame and spliceacceptor, SV2 hygromycin selectable marker, and optionally the mouse 3′enhancer sequence.

DETAILED DESCRIPTION

[0041] Before the methods and compositions of the present invention aredescribed and disclosed it is to be understood that this invention isnot limited to the particular methods and compositions described as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims.

[0042] It must be noted that as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a DNA sequence” includes a plurality of DNAsequences and different types of DNA sequences.

[0043] Unless defined otherwise all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any materialsor methods similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the particular information for which thepublication was cited in connection with. The publications discussedabove are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventor is not entitled to antedate such disclosureby virtue of prior invention.

[0044] Definitions

[0045] The term “artificial” as used with “artificial construct” or“artificial switch region” and the like, refers to an isolated naturalor non-naturally occurring material e.g., a nucleotide sequencemanufactured by human intervention e.g., fusing natural sequencestogether or chemically synthesizing natural sequences in isolation.

[0046] The term “switch region” means a nucleotide sequence composed oftandem repeat sequences that occur in nature 5′, to the immunoglobulinheavy chain constant region and function in intrachromosomalclass-switching, i.e., recombination of DNA sequences encoding specificportions of immunoglobulin heavy chain constant regions. Examples ofspecific switch region sequences are disclosed in Mills et al. (1995) J.Immunol. 155:3021-3036, herein specifically incorporated by reference.“Switch region” includes both full-length switch sequences of nativeimmunoglobulin sequences, as well as recombinant and syntheticnucleotide sequences that are modified (e.g., contain nucleotidesubstitutions, additions, mutations, and/or other modifications)relative to a native immunoglobulin switch region, with the proviso thatthe switch region retains its function in facilitating recombinationwhen transcribed.

[0047] The term “switch-mediated recombination” or “directed S-Srecombination” are used interchangeably to mean interchromosomal,intrachromosomal, or extrachromosomal DNA recombination facilitated by aswitch region. For example, S-S recombination results from interactionof 1) a first switch region positioned 3′ to a promoter (targetingconstruct) and 2) a second switch region positioned 3′ to a promoter and5′ to a DNA sequence (target locus). Following activation oftranscription of the first and second switch regions, recombinationoccurs between the switch regions resulting in an alteration of thetarget locus DNA sequence. The directed S-S recombination of theinvention results in interaction between DNA sequences on two differentchromosomes, on the same chromosome, between a chromosome and anextrachromosomal element, or between two extrachromosomal elements.

[0048] The term “targeting construct” means a nucleic acid constructwhich is introduced into a cell to cause directed S-S recombination at anatural or artificial switch region. A targeting construct minimallycomprises: 1) a switch region and 2) a promoter operably linked to and5′ of the switch region. Optionally, the targeting construct furthercomprises 3) a modifying sequence operably linked to and 3′ of theswitch region. The targeting construct may also comprise 4) one or moreDNA sequences between the switch region and promoter. Depending on theactual targeting construct used, the resulting mRNA will encode theswitch region, or the switch region and the modifying sequence, or theswitch region and DNA sequences between the switch region and/or amodifying sequence.

[0049] The term “target locus” means a nucleic acid sequence minimallycomprises 1) a switch region, 2) a target sequence adjacent and 3′ ofthe switch region, and 3) a promoter operably positioned in the targetlocus to provide transcription of the switch region and target sequenceas one or more translatable mRNA(s). The target locus can furthercontain an additional DNA sequence positioned adjacent and 5′ of theswitch region; in such constructs, the promoter provides transcriptionof the additional DNA sequence, the switch region, and the targetsequence as one or more translatable mRNA(s). “Target loci” can beeither naturally occurring (e.g., an immunoglobulin gene composed of arearranged VDJ region positioned 5′ of a switch region and C_(H)gene) orrecombinantly or synthetically produced, and can be either chromosomalor extrachromosomally located. An exemplary target sequence comprises apromoter sequence operatively positioned 5′ to a switch regionoperatively positioned 5′ to a coding sequence which is preferably asequence encoding a constant region of a human antibody.

[0050] The terms “target sequence” means the nucleic acid sequenceadjacent to a switch region where directed S-S recombination takesplace. In one embodiment of the method of the invention, a targetsequence is replaced by the modifying sequence after switch-mediatedrecombination. “Target sequences” can be naturally occurring sequencesendogenous to a chromosomal sequence or recombinant sequences (i.e., asequence produced using recombinant genetic manipulation) present as anextrachromosomal element (e.g., a vector) or as a stably integratedelement within a chromosomal sequence. Target sequences are adjacent toa switch region which may be a naturally occurring switch region or maybe a switch region inserted 5′ to a desired target sequence byrecombinant DNA technology. Exemplary target sequences are differentfrom the modifying sequence and include sequences encoding animmunoglobulin heavy chain constant region of a particular isotype,subtype, and/or origin.

[0051] The term “immunoglobulin (Ig) locus” means a nucleotide sequencethat encodes all or a portion of the constant region and/or variableregion of an antibody molecule, including all or portions of theregulatory sequences that control expression of an antibody moleculefrom the locus or its processes. Heavy chain genes in Ig loci includebut are not limited to all or a portion of the V_(H), D_(H), J_(H), andconstant regions, as well as the switch regions, intronic sequences, andflanking sequences associated with or adjacent the heavy chain gene. Igloci for light chains include but are not limited to the V_(L), J_(L),and constant regions of both the kappa and lambda alleles, intronicsequences, and flanking sequences associated with or adjacent the lightchain gene.

[0052] The term “modified target locus” means a nucleic acid sequencemodified by switch-mediated DNA recombination so that the modifiedtarget sequence is minimally composed of a switch region composed ofswitch sequences derived from the unmodified target locus switch region,or from both the unmodified target locus and the targeting construct. Inone embodiment of the invention, the modified target locus is alsocomposed of the promoter of the unmodified target sequence, the firstDNA sequence of the unmodified target sequence (when present in theoriginal target locus), and the modifying sequence of the targetingconstruct. Activation of transcription by the promoter results intranscription of the first DNA sequence, the switch region, and themodifying sequence in one or more translatable mRNA(s).

[0053] The term “promoter” means a nucleotide sequence that, whenoperably linked to a DNA sequence of interest, promotes transcription ofthat DNA sequence.

[0054] The term “detectable polypeptide label” means a amino acidsequence that, when covalently bound to another amino acid sequence,provides a heterologous sequence that can be readily detected. Forexample, the polypeptide can be detected by binding of apolypeptide-specific antibody, by virtue of an enzymatic activity of thepolypeptide, or by reaction of the polypeptide with a chemical reagent.Exemplary detectable polypeptide labels include β-galactosidase,alkaline phosphatase, horseradish peroxidase, enzymatically activeportions of these enzymes, or any amino acid sequence that isimmunodetectable and heterologous to the amino acid sequence with whichit is associated.

[0055] Directed S-S Recombination (General)

[0056] The method of directed switch region-mediated recombination usesswitch regions (e.g., those isolated and derived from an immunoglobulinlocus) to facilitate recombination at a specific nucleic acid sequence.The nucleic acid sequence to which S-S recombination is directedcontains an S region and is termed a “target locus,” while theintroduced nucleic acid sequence containing a S region sequence istermed a “targeting construct.” Transcription of each S region allowsS-S recombination to occur between the two preselected DNA regions. Thepresence of a selected promoter provides constitutive or inducibletranscription, thereby enhancing the frequency of S-S recombinationoccurrence.

[0057] The basic components of an exemplary target locus suitable foruse in the invention are illustrated in FIG. 2A. The minimal componentsof the target locus are (from 5′ to 3′): 1) a promoter (P₂, where thearrow indicates the direction of transcription), 2) a switch region(S₂), and 3) a target sequence (T). Alternatively, the target locus canfurther contain an additional DNA sequence positioned 3′ of the promoterand 5′ of the switch region (Y) (FIG. 2B). Regardless of itscomposition, the target locus components are positioned so that thepromoter activates transcription of the 5′ DNA sequence (optional),switch region, and target sequence (optional). The target locus caneither be an endogenous, naturally-occurring chromosomal sequence (e.g.,an Ig heavy chain locus where the 5′ DNA sequence is a V_(H)D_(H)J_(H)gene and the target sequence is a C_(H) gene) or an artificiallyconstructed sequence (i.e., a recombinantly produced sequence or asynthesized sequence) which is present as either an extrachromosomalelement (e.g., a vector or plasmid) or as a stable chromosomalintegrant.

[0058] The basic components of an exemplary targeting construct for usein the invention are illustrated in FIGS. 3A-3D. The minimal componentsof the targeting construct are (from 5′ to 3′): 1) a promoter (P₁ wherethe arrow indicates the direction of transcription) and 2) a switchregion (S) (FIG. 3A). The targeting construct can additionally contain3) a modifying sequence 3′ to S (FIG. 3B), and/or 4) one or more DNAsequences 5′ to S (FIG. 3C). Additionally, the targeting construct caninclude a selectable marker (FIG. 3D). The targeting constructcomponents are positioned so that the promoter activates transcriptionof the switch regions and modifying sequence. The targeting construct isnormally a recombinantly or synthetically produced nucleic acidsequences, and can be used in the method of the invention as either anextrachromosomal element (e.g., a plasmid or vector) or as a stablechromosomal integrant. Exemplary modifying sequences include the C_(H)gene for use in isotype switching (i.e., replacement of the C_(H) geneof the target locus with a C_(H) gene of a different isotype orsubtype).

[0059] The precise mechanism through which intrachromosomal S-mediatedrecombination (also termed S-S recombination) occurs in the class-switchphenomenon is not fully understood (for a review on this topic, seeCoffman et al., 1993, Adv. Immunol. 54:229-71). Without being held to aspecific theory, naturally-occurring S-mediated recombination istriggered by simultaneous transcription of two intrachromosomal switchregions (Xu & Stavnezer (1990) Develop. Immunol. 1:11-17; Rothman et al.(1990) Mol. Cell Biol. 10:1672-1679; Jung et al. (1993) Science159:984-987). For example, in a cell producing IgM antibody, the IgMheavy chain gene (which includes a V_(H)D_(H)J_(H) region, a switchregion (Sμ), and a Cμ gene) is constitutively transcribed andtranslated. Class-switching (e.g., to production of IgG) occurs when asecond switch region (e.g., Sγ) is transcribed. Transcription of asecond switch region is thought to be regulated by control elementsassociated with each of the switch regions of the C_(H) locus. Each ofthese control elements are activated by a different combination ofcellular signals (i.e., one or more cellular signals) normallyassociated with cytokines which can be activated, for example, in amicrobial infection or inflammation (e.g., cytokines such asinterleukins, interferons, and tumor necrosis factor). In turn,production of cellular signals is associated with specific types ofinfections and inflammation. Thus, a specific type of infection orinflammation results in: 1) production of a specific combination ofcellular signals, which in turn determines 2) which of the switch regioncontrol elements is activated and, as a result, 3) which switch regionis transcribed to promote recombination of its associated C_(H) regionwith the constitutively transcribed Sμ and Cμ regions to produce adifferent, specific antibody isotype (Coffman et al., 1993, supra).

[0060] The present invention uses switch regions to provide a method ofdirecting recombination to pre-selected sites of interest in a mannerthat is not controlled by the normal cellular regulatory mechanismsdescribed above. As illustrated in FIGS. 4A-4C, the directed S-Srecombination of the present invention uses a targeting constructminimally containing a switch region (S₁) and a promoter (P₁), and atarget locus containing a switch region (S₂) and target sequence (T)under control of a promoter (P₂), to facilitate switch-site specificrecombination mediated by the two transcriptionally activated switchregions. The resulting recombinatorial product will minimally contain aswitch region having sequences from one or both switch regions, e.g.,S₁, or S₁/S₂. When the targeting construct contains a promoter P₁ andS₁, the desired recombinatorial product will consist of the P₁ promoter,the switch region, and the target sequence, now under control of P₁instead of P₂ (FIG. 4A). The desired recombinatorial product isrecognized in a number of ways known to the art including PCR. When P₁is an inducible promoter, a cell containing the desired recombinatorialproduct can be recognized by induction of transcription. When thetargeting construct consists of P₁, S₁, and a modifying sequence, thedesired recombinatorial product will consist of the P₂ promoter, theswitch region, and the modifying sequence which replaces the targetsequence (FIG. 4B). The switch region may contain sequences from one orboth switch regions, e.g., S₁ or S₁/S₂. When the modifying sequenceencodes a protein or peptide, the desired recombinatorial product can berecognized by synthesis of the desired product. When the targetingconstruct consists of P₁, DNA sequences 5′ to S₁, and S₁, the desiredrecombinatorial product contains P₁ and the DNA sequences 5′ to the S₁region inserted into the target locus (FIG. 4C). The desiredrecombinatorial product can be identified in a variety of ways,including PCR detection of the presence of the 5′ DNA sequences and/orP₁, or by immunodetection technologies.

[0061] Additionally, the targeting construct can be used to insert apiece of DNA 3′ to a target locus contained in a specific chromosome. Inthis embodiment, the targeting construct carries homologous sequencesallowing insertion into the selected chromosome by homologousrecombination. The resulting modified chromosome contains a DNA of thetargeting construct at a site 3′ from the target locus. This embodimentis useful for induction of intrachromosomal S-mediated recombination.

[0062] Switch Regions

[0063] Class-switching (or isotype switching) results when B lymphocytesinitially expressing IgM switch their heavy chain isotype to IgG, IgA,or IgE upon maturation. Isotype switching results from a deletional DNArecombination event in which the Cμ constant region of the heavy chain,initially located downstream of the V_(H)D_(H)J_(H) region, is replacedby a C_(γ), C_(α), or C_(ε), constant regions (Rabbitts et al. (1980)Nature 283:351; Davis et al. (1980) supra; Kataoka et al. (1981) supra.

[0064] Several switch regions have been characterized, including themurine Sμ, Sε, Sα, Sγ₃, Sγ₁, Sγ_(2b) and Sγ_(2a) switch regions and thehuman Sμ switch region, such as S_(μ), and S_(γ4) (Mills et al. (1995)J. Immunol. 155:3021-3036, herein specifically incorporated byreference). The murine Sμ region is about 3 kb and can be divided into a3′ region with sequences of [C(GAGCT)nGGGGT]m, where n=1-7 and m=150(Nikaido et al. (1981) supra), and a 5′ region in which these twopentamers are interspersed with the pentamer sequence (C/T)AGGTTG (Marcuet al. (1982) supra) . The human Sμ locus is slightly different in thatthe heptamer sequence is distributed throughout the region (Takahashi etal. (1982) supra; Mills et al. (1990) supra). Although other switchregions contain more complex patterns of repeated sequence, all switchsequences contain multiple copies of the pentameric sequences GAGCT andGGGGT (Nikaido et al. (1982) supra; Stanton et al. (1982) supra). Thepentamers ACCAG, GCAGC, and TGAGC are also commonly found in switchregions (Gritzmacher (1989) supra). In addition, the heptameric repeat(C/T)AGGTTG is abundantly present in switch region sequences and isfound near many, but not all, switch recombination sites that have beencharacterized in plasmacytomas and hybridomas (Marcu et al. (1982)supra).

[0065] The murine Sε and Sα loci contain 40 bp and 80 bp sequences,respectively, that are tandemly repeated. These sequences are homologousto Sμ, especially in areas of the repeats containing the GAGCT pentamer.Both human and murine Sγ regions are much less homologous to Sμ than arethe Sε and Sα regions. The homology of murine Sγ regions to Sμ decreaseswith the increasing distance 3′ of the variable region(Sγ3>Sγ1>Sγ2b>Sγ2a). The murine Sγ regions are composed of tandemrepeats of 49 bp or 52 bp (Sγ2a), within which the pentameric sequencesTGGGG, GCAGC, and ACCAG are commonly found (Kataoka et al. (1981) supra;Mowatt et al. (1986) supra; Nikaido et al. (1982) supra, Nikaido et al.(1981) supra; Stanton et al (1982) supra; Szurek et al. (1985) supra; Wuet al. (1984) supra).

[0066] Switch regions suitable for use in the invention can be naturallyoccurring sequences, e.g., a switch region cloned directly from an Iglocus, preferably from a murine or human Ig locus. Alternatively, theswitch region can be a synthetically or recombinantly produced sequence.Recombinant switch regions can have the same sequence as a native,naturally-occurring switch region, or can be modified (e.g., containnucleotide substitutions, additions, mutations, and/or othermodifications) relative to a native switch region, with the proviso thatthe switch region retains its function in facilitating recombination.Recombinant switch regions can be designed to as to have a minimalnucleotide sequence necessary for switch-mediated recombination at thesame (or lower but acceptable) level as a native switch region, or at alevel enhanced relative to recombination promoted by a wild-type switchregion.

[0067] The switch-mediated recombination of the present inventionprovides improved efficiency of S-S recombination over thenaturally-occurring mechanism, as well as providing a widely applicationmethod of producing a desired protein. This is achieved, in part withthe use of promoters providing constitutive or inducible transcriptionof the targeting construct, the target locus, or both the targetingconstruct and target locus. The improved efficiency of theswitch-mediated recombination method of the invention provides afrequency of recombination at a level higher than that which occursnaturally, that is, a 1% to 100% improved efficiency; more preferably, a20% to loot improvement; and more preferably a 50% to 100% improvement.

[0068] Targeting Constructs

[0069] As discussed above, targeting constructs of the invention areminimally composed of: 1) a switch region and 2) a promoter operablylinked to and 5′ of the switch region. Additional optional components ofthe targeting construct include 3) a modifying sequence operably linkedto and 3′ of the switch region, including proteins, selectable markers,and/or control elements, and/or 4) DNA sequences 3′ to the promoter and5′ to the switch region. Transcriptional activation of the promoterresults in production of one or more translatable mRNA(s)).

[0070] The Targeting Construct Promoter

[0071] The promoter of the targeting construct is selected according tothe cell type in which directed S-S recombination is to be accomplished(e.g., a eukaryotic or prokaryotic cell, normally a eukaryotic cell).Because directed S-S recombination is dependent on transcription of theswitch regions of the targeting construct and the target locus, thepromoter of the targeting construct can be a constitutive or aninducible promoter. Suitable constitutive and strong constitutivepromoters for DNA expression in prokaryotic or eukaryotic cells are wellknown in the art. Where the cell in which directed S-S recombination isto take place is a eukaryotic cell, the promoter can be the heavy chainIg promoter or a viral promoter, such as a CMV, SV40, murine MoloneySarcoma virus (MMLV), and spleen-focus forming virus (SFFV) promoter, oran inducible promoter, such as MMTV and α-inhibin.

[0072] The Modifying Sequence

[0073] The modifying sequence can be any nucleic acid sequence that issuitable for replacing a target sequence in a target locus. For example,the modifying sequence can be composed of a nucleotide sequence thatencodes a translation product to replace all or a portion of the targetsequence. For example, where the target sequence is a C_(H) gene, themodifying sequence can be a different native C_(H) gene, a modifiedC_(H) gene (e.g, encoding an altered effector function relative to thewild-type C_(H) gene), or a native or modified light chain constantregion. Alternatively, the modifying sequence can encode anon-antibody-derived polypeptide that confers a function upon thepolypeptide encoded by the modified target sequence. For example, themodifying sequence can encode a toxin, hormone, growth factor, orportions thereof. The modifying sequence can also encode a linker toprovide covalent or non-covalent linkages between other (e.g., similarlymodified) heavy chain gene products or non-antibody polypeptides (e.g.,toxins, growth factors, hormones, or other biologically importantpolypeptide or other molecule). Yet another example of a modifyingsequence is a nucleotide sequence encoding a detectable polypeptidelabel or tag, e.g., β-galactosidase, alkaline phosphatase, horseradishperoxidase, or an immunodetectable polypeptide to which an antibody canbind to facilitate polypeptide detection and/or isolation (e.g., byimmunoaffinity chromatography).

[0074] Alternatively or in addition, the modifying sequence can containregulatory sequences (e.g., a promoter, enhancer element, an intron, ora ribosome binding site) that can be used to either introduce regulatorysequences at a position 3′ of a switch region, or to replace regulatorysequences already present in the target sequence. For example,switch-mediated recombination can be used to replace a weak promoterwith a strong promoter in a target locus, where the weak promoter ispositioned 3′ or 5′ of the target locus switch region. Exemplaryregulatory sequences of particular interest in the modification of an Iglocus include a heavy chain enhancer sequence, a kappa chain enhancersequence, or a promoter derived from MMLV, Rous sarcoma virus (RSV), orSFFV.

[0075] The targeting construct may also contain an amplification genethat allows the modified target locus to be amplified switch-mediatedproduct. There are a number of suitable amplification genes known to theart and useful in the invention, for example, the gene encodingdihydrofolate reductase (DHFR).

[0076] The modifying sequence is selected according to a variety offactors including the target sequence to be modified, and/or thediagnostic or therapeutic use intended for the resultant recombinatorialproduct.

[0077] Additional Sequences Present 3′ of the Promoter and 5′ of theSwitch Region

[0078] The targeting construct can contain an additional, transcribableand translatable DNA sequence operably positioned between the promoterand switch region of the target locus. This additional sequence canencode an N-terminal portion of the polypeptide encoded by the targetlocus. For example, the targeting construct can encode a desiredV_(H)D_(H)J_(H) polypeptide. Upon directed switch-recombination with atarget locus encoding an Ig heavy chain locus having a desired C_(H)gene at the target sequence, the recombinatorial product contains thedesired V D_(H)J_(H)region and the desired C_(H) coding region, with theswitch region positioned between.

[0079] Other Components

[0080] The targeting construct can be based upon any of a variety ofvectors that are well known in the art and commercially available (e.g.,pBR322, pACYC vectors, plasmids, and viral vectors). “Vectors” includeany DNA or RNA molecule (self-replicating or not) that can be used totransform or transfect a desired cell. The targeting construct caninclude other components such as a selectable marker to facilitatescreening and selection of cells containing the targeting construct asan extrachromosomal or chromosomally integrated element, and/or toselect for cells that have successfully undergone directed S-Srecombination, e.g., a selectable marker associated with the modifyingsequence that is recombined into the target locus in addition to themodifying sequence. Suitable selectable marker genes include genesencoding a detectable marker (e.g., β-galactosidase) or drug resistancegenes, e.g., hygromycin resistance (hyg), guanosine phosphoryltransferase (gpt), neomycin resistance (neo), dihydrofolate reductase(DHFR), puromycin (spt) and ampicillin resistance (Amp). The constructcan also include an origin of replication for stable replication of theconstruct in a bacterial cell (preferably, a high copy number origin ofreplication), a nuclear localization signal, or other elements whichfacilitate production of the DNA construct, the protein encoded thereby,or both. For eukaryotic expression, the construct may also anamplification gene, which can increase levels of expression of the DNAof interest, particularly where the DNA of interest is a cDNA (e.g.,contains no introns of the naturally-occurring sequence). Any of avariety of amplification genes known in the art may be used, includingDHFR.

[0081] Target for Use with Targeting Constructs

[0082] As discussed above, a target locus suitable for use in the methodof the invention is minimally composed of: 1) a switch region, 2) atarget DNA sequence adjacent and 3′ of the switch region, and 3) apromoter operably positioned in the construct to provide transcriptionof the switch region and target sequence as an mRNA molecule. The targetlocus can further contain an additional DNA sequence positioned adjacentand 5′ of the switch region; in such constructs, the promoter providestranscription of the additional DNA sequence, the switch region, and thetarget sequence as a one or more translatable mRNA(s).

[0083] In general, target loci suitable for use with the targetingconstructs of the invention can be any switch-containing sequence inwhich the switch region is transcribed and can facilitateswitch-mediated recombination. The target locus can be any native,endogenous chromosomal sequence which contains a switch region (e.g., anIg heavy chain locus). Alternatively, the target locus can be anartificially, recombinantly produced sequence present as either anextrachromosomal element (e.g., a vector or plasmid) or a chromosomallyintegrated element. In a specific embodiment of the invention, where itis desirable to insert a portion of a targeting construct 3′ of a targetlocus on the same chromosome, the targeting construct carries homologoussequences directing recombination at a site 3′ of a target locus.S-mediated recombination will then take place intrachromosomally, thusallowing controlled induction of intrachromosomal recombination.

[0084] The Promoter

[0085] The promoter of the target locus (P₂) can be the promoter that ispresent in the native, naturally-occurring target locus sequence and/orthe target sequence, or a promoter that is heterologous to the targetlocus sequence and/or the target sequence. Because S-S recombination isassociated with transcription of the switch region, the target locuspromoter preferably provides at least low-level expression, morepreferably constitutive expression, and even more preferably, provideshigh levels of constitutive expression of the target locus, specificallyof the switch region-encoding DNA. Where the promoter associated withthe target locus provides inadequate levels or undesirably low levels oftranscription of the switch region, the native target locus promoter canbe modified or replaced with a different promoter using S-mediatedrecombination or other recombinant methods well known in the art, e.g.,cloning, homologous recombination).

[0086] Additional Sequences Present 3′ of the Promoter and 5′ of theSwitch Region

[0087] As discussed above, the target locus can contain an additional,transcribable and translatable DNA sequence operably positioned betweenthe promoter and switch region of the target locus. For example, theadditional sequences may encode an N-terminal portion of the polypeptideand the target locus contains a target sequence encoding the C-terminalportion of a polypeptide. After directed switch-mediated recombination,the modified target locus will contain both the N- and C-terminalportions of the polypeptide.

[0088] Other Components

[0089] The target locus can include additional components to facilitatereplication in prokaryotic and/or eukaryotic cells, integration of theconstruct into a eukaryotic chromosome, and markers to aid in selectionof and/or screening for cells containing the construct (e.g., thedetectable markers and drug resistance genes discussed above for thetargeting construct). For eukaryotic expression, the construct shouldpreferably additionally contain a polyadenylation sequence positioned 3′of the gene to be expressed. The polyadenylation signal sequence may beselected from any of a variety of polyadenylation signal sequences knownin the art. Preferably, the polyadenylation signal sequence is the SV40early polyadenylation signal sequence. Expression of the target locuscan also be enhanced by inclusion of intronic sequences, as discussedabove for the targeting construct.

[0090] An exemplary recombinant target locus of the invention iscomposed of (from 5′ to 3′): 1) a promoter, 2) a first multiple cloningsite for insertion of a DNA sequence 5′ to the switch region, 3) aswitch region, and 4) a second multiple cloning site for insertion of atarget DNA sequence. For example, where the target locus is arecombinant Ig heavy chain gene, a V_(H)D_(H)J_(H) DNA sequence isinserted 5′ to the switch region at the first multiple cloning site, andthe target sequence is a C_(H) region inserted into the second cloningsite.

[0091] Cell Lines Suitable for Use with the Method of the Invention

[0092] Any mammalian cell line capable of expressing the target locus ofinterest is suitable for use in the present invention. For example,where the target locus is an Ig heavy chain gene, the cell line is anymammalian cell capable of expressing a functional antibody. Ofparticular interest is the use of the switch-mediated recombinationmethod of the invention to facilitate class-switching inantibody-producing cells or cells with antibody-producing potential(e.g., stem cells). For example, the cell line can be, e.g., a hybridomacell line expressing human antibodies, an embryonic stem cell (e.g., amurine embryonic stem cell), a hybridoma cell line produced from B cellsfrom a transgenic animal (e.g., a transgenic mouse), or any other cell(normally a mammalian cell) capable of expressing at least a functionalportion of a heavy chain Ig locus or at least a functional portion of alight chain Ig locus. One example of a cell line useful in the method ofthe invention is a hybridoma cell line expressing human antibodiesderived from B cells from the Xenomouse (Green et al. (1994) NatureGenetics 7:13 and PCT patent publication No. WO 94/02602, both of whichare herein specifically incorporated by reference). The Xenomousecarries large segments of the human heavy chain and K chain lociintegrated into its germline, as well as containing functionallyinactivated mouse heavy and kappa light chain alleles. Xenomouseproduces B cells expressing human heavy chain (hμ) and human K lightchain (mK), or hμ and mouse lambda (mλ) light chain. Co-expression of hκand mλ does not occur, since expression of one light chain completelyexcludes the expression of the other (Green et al. (1994) supra. Uponimmunization, Xenomouse produces a broad adult-like repertoire of humanIg and give rise to antigen-specific human monoclonal antibodies.Xenomouse allows generation of mouse hybridomas making antigen-specifichuman monoclonal antibodies. Methods for producing hybridoma cell linesare well known in the art (see, for example, Harlow and Lane, eds.,1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Methods for producing cell lines expressinghuman or “humanized” antibodies are also well known in the art (see, forexample, PCT Publication Nos. WO 94/02602 and WO 91/10741).

[0093] Where the cell line is an antibody-producing lymphoid cell line,the cell line can express the antibody from either a genomic sequence, amodified sequence, a heterologous sequence (e.g., an Ig sequence fromanother species), a modified heterologous sequence, or a chimericsequence (e.g., composed of both murine and human Ig sequences). Thus,the cell line can be, for example, a murine hybridoma cell lineproducing either a murine, human, or chimeric antibody. The hybridomacell line can be producing human antibodies by, for example, expressionof human Ig genes. In one embodiment, the cell is a murine lymphoid cellproducing a human antibody by expression of human Ig genes. In onevariation of the embodiment, the constant region gene of the genomicsequence is a human constant (hC_(H)) region gene, e.g., a hC_(H) geneof the mu class (hC_(H)μ), and the modifying sequence is a humanconstant region of the gamma class (hC_(H)γ).

[0094] Methods Using Switch-Mediated Recombination

[0095] Switch-mediated recombination using the construct(s) of theinvention can be accomplished in a variety of ways. For example, 1) thetarget locus can be naturally occurring (chromosomally located) and thetargeting construct can be used as either an extrachromosomal orchromosomally integrated element; or 2) the target locus can be anaturally occurring or recombinantly produced sequence that is eitherpresent as an extrachromosomal or a chromosomally integrated element,and the targeting construct can be used as either an extrachromosomal orchromosomally integrated element. When the targeting construct andtarget locus are both chromosomally integrated, they are integrated onthe same or different chromosomes.

[0096] Switch-Mediated Recombination Using a Chromosomal Target Locusand a Chromosomally Integrated Targeting Construct

[0097] In this embodiment, the cell line used to accomplish directedswitch-mediated recombination either: 1) contains an endogenous,naturally occurring target locus, or 2) contains a chromosomallyintegrated recombinant

locus. Methods for introduction of DNA into a host

selection for stable chromosomal integrants containing a specific DNAsequence of interest are well known in the art (see, for example,Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; herebyincorporated by reference with respect to methods and compositions forrecombinant DNA techniques to provide a transformed cell containing astably integrated DNA of interest, and expression of the DNA ofinterest).

[0098] The targeting construct can be linearized, e.g., by digestionwith a restriction endonuclease(s), and the linear DNA introduced intothe host cell using any of a variety of methods known in the art (e.g.,electroporation, microinjection, liposome fusion, red blood cell ghostfusions, protoplast fusion, yeast cell fusion, or any other method knownin the art (see, for example, Sambrook et al., supra)). The linearvector is then integrated into the cell's genome randomly orspecifically by directed homologous recombination, and stable integrantsare selected by, for example, expression of a selectable markerassociated with the targeting construct, or by expression of themodifying sequence in the targeting construct.

[0099] Directed switch-mediated recombination is accomplished bysimultaneous transcription of the switch regions in the target locus andthe targeting vector. Cells containing the recombinatorial product, forexample, the modified target locus, are identified and selected byexpression of the modified target locus gene product (e.g., by ELISAreactivity or fluorescence-activated cell sorting (FACS)).

[0100] Switch-Mediated Recombination Using a Chromosomal Target Locusand an Extrachromosomal Targeting Construct

[0101] In this embodiment of the method of the invention, the targetingconstruct is introduced into the cell containing a chromosomallyintegrated target locus by methods well known in the art (see, forexample, Sambrook et al., 1989, supra). In contrast to the methodimmediately above, the targeting construct is maintained as anextrachromosomal element for a time sufficient for transcription of thetargeting construct's switch region and recombination with thetranscriptionally active switch region of the target locus. Cellscontaining the desired recombinatorial product, e.g., a modified targetlocus, can be identified and selected as described above, e.g.,selection for expression of a selectable marker associated with theintegrated target sequence, or detection of cells expressing the desiredmodified target locus gene product.

[0102] Screening and Selection

[0103] Detection of properly recombined sequences can be accomplished ina variety of ways, depending upon the nature of the desiredrecombinatorial product. For example, where the modifying sequenceassociated with a selectable marker is recombined into the target locuswith the modifying sequence, an initial screen will select for thosecells which express the marker. A second screen can be used to determineif the drug resistant cells express the appropriately modified targetlocus.

[0104] The method used for the second screen will vary with the natureof the modifying sequence inserted into the target locus. The modifyingsequence can be detected by Southern blot using a portion of themodifying sequence as a probe, or by polymerase chain reaction (PCR)using amplifying primers derived from the modifying and modifiedregions. The cells having an appropriately integrated modifying sequencecan also be identified by detecting expression of a functional modifiedtarget locus product, e.g., immunodetection of the new C_(H) region in amodified antibody heavy chain locus. Alternatively, the expressionproduct of the modified target locus can be detected using a bioassay totest for a particular effector function conferred by the modifyingsequence. For example, the expression of modifying sequence that encodesa biologically active molecule such as an enzyme, toxin, growth factor,or other peptides is assayed for that particularly biological activity.

[0105] Where the target locus is an Ig gene, the product of the modifiedtarget locus can also be tested for appropriate antigen or ligandrecognition via any conventional immunological screening methods knownin the art, e.g, ELISA, FACS, antibody-dependent cell cytotoxicityassays, or immunoprecipitation assays (see, for example, Harlow andLane, supra).

EXAMPLES

[0106] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use various constructs and perform the various methodsof the present invention and are not intended to limit the scope of whatthe inventors regard as their invention. Unless indicated otherwise,parts are parts by weight, temperature is in degrees centigrade, andpressure is at or near atmospheric pressure. Efforts have been made toensure accuracy with respect to numbers used, (e.g., length of DNAsequences, molecular weights, amounts, particular components, etc.) butsome deviations should be accounted for.

Example 1 Targeting Construct for Switch-Mediated Recombination(PTSW-1.4 and pTSW-1-9)

[0107] All the vectors generated were based on a low copy-numberpACYC177 Plasmid (NEB). The vector pTSW-1.4 was generated from- thep1bYACδNot plasmid containing a 23 kb EcoRI genomic fragment of theentire human γ2 switch region, isolated from human placenta genomiclibrary. This fragment contained 2 kb of coding sequences, 12 kb ofupstream sequences including the I exon and γ72 switch region, and 9 kbof downstream sequences (Flanagan & Rabbitts (1982) Nature 300:709-713).This plasmid also contains the mouse 3′ enhancer (Dariavach et al.(1991) Eur. J. Immunol. 21:1499-1504). The vector was modified tocontain a hygromycin selectable marker and a human CMV promoter-enhancercassette, which included at its 3′ end prokaryotic terminator sequences(described below). The prokaryotic terminator sequences were used tostop fortuitous prokaryotic transcripts from activating the switchsequences, and thus destabilizing them during cloning in bacteria(Powatt & Dunnick (1986) J. Immunology 136:2674 -2683). These sequenceswere confirmed to have very little effect on eukaryotic transcription.

[0108] The hygromycin gene, driven by the SV40 promoter (Giordano &McAllister (1990) Gene 88:285-288) was cloned as a 1.7 kb HindIII-BamHIfragment from pUC219.TG76 plasmid and inserted into HindIII and BamHIsites in pACYC177 to generate pACYC.hyg plasmid.

[0109] The terminator was synthesized asGCATGCCCGCGG-GAATAGGCGGGCTTTTTTNNNGCCGCGGCTCGA (SEQ ID NO: 1), withflanking SphI sites, and an internal XhoI site at the 3′ end, forcloning purposes. This sequence was cloned into the SphI site ofpIK1.1Cat plasmid, downstream of the human CMV promoter-enhancersequences.

[0110] The CMV expression cassette, together with the terminatorsequences, was cloned as a 900 bp HindIII-XhoI fragment, which wasplaced into the HindIII and XhoI sites in the pACYC.hyg plasmiddescribed above to generate pACYC.hyg.CMVt, in which the CMVtranscription orientation is opposite to that of the hygromycin gene.

[0111] The 2.6 kb fragment, containing both hygromycin andCMV-terminator cassettes, was excised from pACYC.hyg.CMVt by BamHI andXhoI digestion. Both ends of this fragment were converted into NotIsites, using linkers, and the fragment was cloned into the unique NotIsite in p1bYACδNot plasmid containing 23 kb human γ2 sequences and mouse3′ enhancer. pTSW-1.4 plasmid (FIG. 5) was generated with CMVtranscription orientation in the same direction as that of the human γ2coding sequences. pTSW-1.9 plasmid (FIG. 6) was generated with CMVtranscription orientation opposite to that of the human γ2 codingsequences.

[0112] An exemplary targeting construct of the invention, designatedpTSW-1.4, is shown in FIG. 5. pTSW-1.4 is constructed for use. inswitch-mediated replacement of an Ig heavy chain constant gene with ahuman heavy chain IgG₂ constant gene (hCHγ2). The pTSW-1.4 constructcontains a CMV promoter operably linked to, in the 5′ to 3′ orientation,the human IgG2 heavy chain region (approximately 23 kb), which includesthe IgG2 heavy chain I exon and its 5′ flanking sequences, the humanIgG2 switch region, the complete human hCHγ2 gene, and sequencesflanking the IgG2 heavy chain region. The hCHγ2 region is linked to amurine enhancer positioned adjacent and 3′ of the hCHγ2 gene. The CMVpromoter is a strong constitutive promoter. Other constitutive promoterscan be used instead of the CMV promoter (e.g., SSFV, MMLV, MCV, RSV,SV40, etc.). Both the hCHγ2 regions have been cloned and sequenced(Mills et al. (1995) supra). Murine 3′ enhancer is also well known inthe art (Dariavach et al. (1991) supra).

Example 2 Targeting Construct for Switch-Mediated Recombination (pTSW-2)

[0113] To generate pTSW-2 plasmid, a 13 kb BamHI fragment was clonedfrom the 23 kb EcoRI human γ2 genomic fragment in p1bYACδNot plasmid, asdescribed in Example 1, followed by partial fill-in reaction with Klenowto generate fragment ends compatible with XhoI. This clone was insertedinto the unique XhoI site in pACYC.hyg.CMVt plasmid, which was alsopartially filled in with Klenow to make the site compatible with BamHI.The correct orientation of the clone, in which the transcriptionorientation of human γ2 coding sequences is the same as the CMVpromoter, was selected.

[0114] Another exemplary targeting construct of the invention,designated pTSW-2, is shown in FIG. 7. Like pTSW-1.4, pTSW-2 isconstructed for use in switch-mediated replacement of an Ig heavy chainconstant gene with a human heavy chain IgG₂ constant gene (hCHγ2). ThepTSW-2 construct is prepared using a CMV promoter operably linked to, inthe 5′ to 3′ orientation, the human IgG2 heavy chain region(approximately 13 kb) including the human IgG2 switch region and 200 bp5′ flanking sequences of the switch region and human γ2 open readingframe. Some of the flanking sequences present in pTSW-1.4 are notpresent in pTSW-2. The pTSW-2 construct also contains the selectablemarker SV2hyg and a prokaryotic transcription terminator (to stabilizethe switch region). The pTSW-2 construct can be prepared either with orwithout a murine enhancer positioned 3′ of the hCHγ2 gene.

Example 3 Targeting Construct for Switch-Mediated Recombination(pTSW-3.1)

[0115] To generate pTSW-3.1 plasmid (FIG. 8), 2 kb of human γ2 codingsequences were cloned by PCR from p1bYACδNot as an XhoI-SalI fragment(Example 1). This fragment was cloned into the unique XhoI inpACYC.hyg.CMVt plasmid, 3′ of the terminator sequences. Mouse γ1 switchsequences were excised as a 10 kb HindIII-EcoRI fragment fromp-gamma-1/EH10.0 plasmid (Mowatt & Dunnick (1986) supra), and the endswere converted into XhoI and SalI, respectively. The modified plasmidwas cloned 5′ of the human γ2 region via the unique XhoI site inpACYC.hyg.CMVt. pTSW-3.1dBglII plasmid (FIG. 9) was generated similarlyto pTSW-3.1, except that a 7.9 kb BglII-EcoRI mouse γ1 switch sequenceswas included.

[0116] pTSW-3.2 was constructed as described for pTSW-3.1, except thatthe CMV promoter-enhancer cassette was replaced by the spleen focusforming virus (SSFV) promoter.

[0117] pTSW-3 plasmids contained unique NotI and MluI sites (convertedby a linker from the unique BamHI site). HindIII is used forlinearization and for cloning of the 3′ enhancer.

[0118] A further exemplary targeting construct of the invention,designated pTSW-3.1, is shown in FIG. 8. Like pTSW-1.4 and TSW-2,pTSW-3.1 is constructed for use in switch-mediated replacement of an Igheavy chain constant gene with a human heavy chain IgG₂ constant gene(hCH_(γ2)). The pTSW-3.1 construct is prepared using a CMV promoteroperably linked to, in the 5′ to 3′ orientation, a murine γ1 switchregion which may contain also the mouse γ1 I exon and flankingsequences, and a human genomic constant hCH_(γ1), hCH_(γ2), or hCH_(γ4)coding sequence, which includes the 5′ flanking branch point and spliceacceptor. The pTSW-3.1 construct can optionally further contain a murineγ1 control element (mI_(γ1)) positioned adjacent and 5′ of the mS_(γ1)sequence. Alternatively, the human switch region of the γ1 gene(hS_(γ1)) and its 5′ flanking sequences, such as the I exon (hI_(γ1))can be used instead of the mI_(γ1) and mS_(γ1). In constructs which donot contain I exon, a splice donor site is provided 3′ of the promotersequences. The pTSW-3.1 construct may further optionally contain amurine 3′ enhancer positioned adjacent and downstream of the hC_(Hγ)gene and/or a 3′ eukaryotic transcription terminator positioned 3′ andadjacent the hC_(Hγ) gene. Each of the elements of the pTSW-3 constructare well known in the art (mouse S_(γ4), S_(μ), Mills et al. (1991)supra; mouse S_(γ1), Mowatt & Dunnick (1986) supra; human Sγ, Mills etal. (1995) supra; mouse 3′ enhancer, Dariavach et al. (1991) supra). ThepTSW-3.1 construct also contains the selectable marker SV2 γ2hyg and aHindIII linearization site. Other selectable markers may be used, forexample, pyromycin.

Example 4 Switch-Mediated Recombination in a Hybridoma Cell Line

[0119] As discussed above, one of the problems associated withproduction of human monoclonal antibodies is that the immortal cell linefused with the human B cells is of murine origin. This can result in arecombinatorial event where the resulting antibody has a human variableregion (human light chains and human heavy chain variable region(hV_(H)D_(H)J_(H))), but has a murine heavy chain constant region(mC_(Hγ)) (see FIG. 8). Switch-mediated recombination is used to replacethe mC_(Hγ) gene with a human heavy chain constant region (hC_(Hγ))gene.

[0120] A hybridoma cell line expressing a monoclonal IgG antibodyagainst antigens, including human antigens, having a human heavy chainvariable region (hV_(H)D_(H)J_(H)) and a murine heavy chain constantregion (mC_(Hγ)) is produced using methods well known in the art (forexample, see, Green et al. (1994) supra). A targeting constructcontaining a promoter operably linked to a switch region and the hC_(Hγ)gene is constructed as described above. Any of the exemplary vectorsdescribed in the examples above (pTSW-1.4, pTSW-2, or pTSW-3.1) issuitable for use in this method. The construct is linearized, and thelinear construct introduced into the hybridoma cell by, for example,electroporation, lipofection, or other methods known to the art. Thetransfected hybridoma cells, containing stable integrants of theconstruct, are selected by their ability to grown in hygromycin.Hygromycin resistant cells are then cultured further to allow fortranscription of the target construct from the CMV promoter and theresulting switch-mediated recombinatorial event. Hybridoma single cellcultures are then screened for expression of hC_(Hγ2) by amplificationof recombined antibody message or by using an anti-human IgG2 antibodyin a sandwich ELISA assay, or isolated by FACS sorting.

Example 5 Switch-Mediated Recombination in a Transgenic Mouse ProducingHuman Antibodies

[0121] Switch-mediated recombination may be accomplished in a transgenicmouse in vivo as follows. The targeting vector is introduced as atransgene into a human antibody-producing mouse and the recombinedantibodies, produced by mouse B cells or their derived hybridomas, arescreened as described.

[0122] The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that modifications will occur toone skilled in the art upon reading this disclosure.

1 1 1 45 DNA Artificial Sequence Prokaryotic terminator sequence 1gcatgcccgc gggaataggc gggctttttt nnngccgcgg ctcga 45

What is claimed is:
 1. An artificial nucleic acid targeting constructcomprising: a) a switch region; and b) a promoter operably linked to and5′ of the switch region.
 2. The targeting construct of claim 1 furthercomprising a modifying sequence operably linked to and 3′ of the switchregion.
 3. The targeting construct of claim 1, further comprising anadditional DNA sequence, said additional DNA sequence positioned 5′ ofthe switch region and 3′ of the promoter.
 4. The targeting construct ofclaim 2, wherein the modifying sequence encodes a constant region of anantibody heavy chain.
 5. The targeting construct of claim 4, wherein theconstant region is the constant region of a human antibody heavy chain.6. The targeting construct of claim 4, wherein the modifying sequenceencodes a constant region of a murine antibody heavy chain.
 7. Thetargeting construct of claim 4, wherein the constant region is selectedfrom the group consisting of Cγ, Cμ, Cα, Cδ, and Cε.
 8. The targetingconstruct of claim 2, wherein the modifying sequence encodes animmunoglobulin variable region, a promoter, an enzyme, a toxin, ahormone, a growth factor, a detectable peptide label, or a linkerpolypeptide.
 9. The targeting construct of claim 1, wherein the promoteris a constitutive or inducible promoter.
 10. The targeting construct ofclaim 9, wherein the promoter is a cytomegalovirus promoter, aspleen-focus forming virus (SFFV), a Rous sarcoma virus promoter, andSV40 promoter or a murine Maloney virus promoter.
 11. A cell comprisingthe targeting construct of claim
 1. 12. A cell line comprising a genomewhich has been subjected to directed switch-mediated recombination viaan exogenous nucleic acid targeting construct, said targeting constructcomprising a switch region and a promoter operably linked to and 5′ ofthe switch region.
 13. The cell line of claim 12, wherein said targetingconstruct further comprises a modifying sequence operably linked to and3′ of the switch region.
 14. The cell line of claim 13, wherein saidmodifying sequence encodes a constant region of an antibody heavy chain.15. The cell line of claim 14, wherein said antibody heavy chain is ahuman antibody heavy chain.
 16. A recombinant protein produced by a cellline, said cell line comprising a genome which has been subjected todirected switch-mediated recombination via an exogenous nucleic acidtargeting construct, said targeting construct comprising a switch regionand a promoter operably linked to and 5′ of the switch region.
 17. Theprotein of claim 16, wherein said targeting construct further comprisesa modifying sequence operably linked to and 3′ of the switch region. 18.The protein of claim 17, wherein said modifying sequence encodes aconstant region of an antibody heavy chain.
 19. The cell line of claim18, wherein said antibody heavy chain is a human antibody heavy chain.20. A method for directed switch-mediated recombination, the methodcomprising the steps of: a) introducing a targeting construct into acell, wherein the targeting construct comprises a switch region, and apromoter (P₁) operably linked to and 5′ of the switch region, andwherein the cell comprises a target locus comprising a switch region, atarget sequence adjacent and 3′ of the switch region, and a promoter(P₂) operably positioned within the target locus to providetranscription of the switch region and target sequence; and b) culturingthe cell to allow transcription of the target locus and the targetingconstruct, thereby promoting recombination between the target locusswitch region and the targeting construct switch region; and c)selecting a cell comprising a modified target locus comprising thetargeting construct promoter P₁, a switch region, and the targetingconstruct, wherein P₁, the switch region, and the target sequence areoperably linked and wherein the target sequence is under control of P₁.21. A method for directed switch-mediated recombination, the methodcomprising the steps of: a) introducing a targeting construct into acell, wherein the targeting construct comprises a switch region, and apromoter (P₁) operably linked to and 5′ of the switch region, and amodifying sequence operably linked to and 3′ of the switch region,wherein the cell comprises a target locus comprising a switch region, atarget sequence adjacent and 3′ of the switch region, and a promoter(P₂) operably positioned within the target locus to providetranscription of the switch region and target sequence; and b) culturingthe cell to allow transcription of the target locus and the targetingconstruct, thereby promoting recombination between the target locusswitch region and the targeting construct switch region; and c)selecting a cell comprising a modified target locus comprising thetargeting construct promoter P₂, a switch region, and the modifyingsequence, wherein P₂, the switch region, and the modifying sequence areoperably linked.
 22. The method of claim 21, wherein the target locusencodes an antibody heavy chain.
 23. The method of claim 21, wherein thetarget locus encodes a murine antibody heavy chain and the modifyingsequence of the target construct is a human antibody heavy chainconstant region.
 24. The method of claim 21, wherein the target locusfurther comprises a non-target sequence positioned between the targetlocus promoter and the target locus switch region.
 25. The method ofclaim 24, wherein the non-target sequence encodes a human antibody heavychain variable region, the target sequence encodes a murine antibodyheavy chain constant region, and the modifying sequence of the targetingconstruct is a human antibody heavy chain constant region.
 26. Themethod of claim 21, wherein the targeting construct is linearized priorto step a), and a cell containing a stable integrant of the targetingconstruct is selected prior to step b).
 27. The method of claim 21,wherein the targeting construct is extrachromosomal.
 28. A method for-producing an antibody by switch-mediated recombination, the methodcomprising the steps of: a) introducing a targeting construct into anantibody-expressing cell, wherein the targeting construct comprises aswitch region (S₂), a promoter operably linked to and 5′ of the switchregion, and a modifying sequence operably linked to and 3′ of the switchregion, and wherein the antibody heavy chain expressed by the cell isencoded by an antibody heavy chain locus comprising a promoter, anantibody heavy chain variable region operably linked to and 3′ of thepromoter, a switch region (S₂) adjacent and 3′ of the variable region,and an antibody heavy chain constant region adjacent and 3′ of theswitch region, b) culturing the cell to allow antibody expression andtranscription of the targeting construct, wherein switch-mediatedrecombination between said S₁ and S₂ is promoted and wherein said heavychain locus constant region is replaced with the modifying sequence ofthe targeting construct; and c) selecting a cell comprising a modifiedheavy chain locus, the modified heavy chain locus comprising the heavychain locus promoter, the heavy chain variable region, a switch region,and the modifying sequence of the targeting construct, wherein the heavychain locus promoter, the variable region, the switch region, and themodifying sequence are operably linked.
 29. A nucleic acid target locuscomprising: a switch region, a first cloning site for insertion of anon-target sequence, the first cloning site being adjacent and 5′ of theswitch region, a second cloning site for inserting of a target sequence,the second cloning site being adjacent and 3′ of the switch region, anda promoter operably linked to and 5′ of the first cloning site, whereintranscriptional activation of the promoter provides transcription of thenon-target sequence, the switch region, and the target sequence.
 30. Theconstruct of claim 29, wherein the switch region, first cloning site,second cloning site and promoter are operatively linked in a plasmid.